Translational Control of T4 Phage Gene Expression

本项目的总体目的是阐明T4噬菌体regA基因编码的翻译抑制蛋白在感染大肠杆菌宿主中的生物学作用和作用机制。regA蛋白选择性地抑制噬菌体诱导的mrna子集的翻译，包括其自身的信息（自生翻译抑制）。在过去的几年里，我们克隆了regA基因，过量生产了它的蛋白产物，纯化了蛋白质，纯化了蛋白质，并表征了它在体内和体外的一些活性。我们的一些主要结果包括：regA基因的精确转录定位，自控制靶定位到mRNA的翻译起始区，regA蛋白在几种regA调控mRNA上的结合位点定位，regA蛋白在regA基因下游转录终止中的作用的发现，并发现了一种增强翻译的mRNA结构。在这里提出的工作中，我们将研究regA蛋白mRNA前导段的核苷酸序列在控制该蛋白合成中的作用。regA蛋白结合的几个mRNA靶点（包括自体靶点）正在通过使用体外操作精确定位，通过在regA调节的mRNA的核糖体结合域的特定序列中进行核苷酸替换。这些体外产生的突变对regA蛋白敏感性的影响正在通过体内和体外翻译/抑制试验进行评估。此外，我们正在研究regA蛋白在顺反子区“CUUCGG”发夹结构的转录终止中的作用，并试图确定regA蛋白抑制的mrna的分解产物。一项初步研究正在进行中，旨在确定与regA蛋白相互作用并影响其体内活性的蛋白质、核蛋白和RNA种类。这项研究为我们提供了对细胞控制和协调DNA、RNA和蛋白质合成的整体速率的更深入的了解。

The overall objective of this project is to elucidate the biological roles and mechanism of action of a translational repressor protein that is encoded by the regA gene of bacteriophage T4 in infected Escherichia coli host. The regA protein selectively inhibits translation of a subset of phage-induced mRNAs including its own message (autogenous translational repression). In previous years we cloned the regA gene, overproduced its protein product, purified the protein, purified the protein, and characterized some of its activities in vivo and in vitro. Some of our main results include: precise transcriptional mapping of the regA gene, localization of the target for autogenous control to the translation initiation region of the mRNA, localization of regA-protein-binding sites on several regA-regulated mRNAs, discovery of a role for regA protein in transcription termination downstream of the regA gene, and discovery of an mRNA structure that enhances translation. In the work proposed here we will investigate the roles of nucleotide sequences in the leader segment of the mRNA for regA protein in control of synthesis of the protein. Several mRNA target sites for regA protein binding (including the autogenous target) are being mapped precisely by the use of in vitro manipulations by which nucleotide replacements are made in specific sequences within the ribosome-binding domains of the regA regulated mRNAs. The effects of these in vitro- generated mutations on sensitivity to regA protein are being assessed by in vivo and in vitro translation/repression assays. In addition we are examining the role of regA protein in transcription termination at "CUUCGG" hairpin structures in intercistronic regions and attempting to identify the breakdown products of the mRNAs that the regA protein represses. A pilot study is underway aimed at identifying proteins, nucleoproteins, and RNA species that interact with regA protein and influence its activity in vivo. The work in this proposal should provide us with deeper insights into the as yet poorly understood processes by which a cell controls and coordinates its overall rates of DNA, RNA, and protein synthesis.